Ricin and Shiga toxin are among the most potent poisons known. They are able to kill mammalian cells by catalytically inactivating ribosomes. This entails binding to cell surface receptors, endocytic uptake, and intracellular trafficking to reach a compartment.from which the catalytic fragment translocates into the cytosol where its substrate is located. Because of this, they pose a serious threat to human health, a threat recently extended by their potential use as agents for bioterrorism. At the moment, no effective inhibitors or vaccines are available to combat this danger. The proposed program is a combination of four complementary research projects aimed at overcoming these limitations: a) Developing vaccines. Current vaccination approaches may be limited in the case of ricin because they involve modification of the active site. Our innovative strategies circumvent this problem. b) Transcriptomics and gene traps. Toxins have effects on target organisms that go beyond the inhibition of protein synthesis. We propose to identify genes whose expression is modulated during the early events of intoxication and to screen for genes whose products are necessary for toxin entry and action but non-essential for cell viability. c) Screening for inhibitors. We have developed novel mammalian cell-based high-throughput approaches for identifying inhibitors of ricin and Shiga toxin intoxication, which will be used for screening siRNA and small molecule libraries. Inhibitors identified in this way will be evaluated and further developed as anti-toxin drugs. continued ZAI1GB-M(M1) 4 1 U19 AI061334-01 LORD, J d) Key biological processes. Toxins exploit key cellular processes during intoxication, such as intracellular transport, signaling, and apoptosis. A comprehensive molecular characterization of the role of the genes and inhibitors identified throughout this project may likewise produce further potential targets for therapeutic intervention. Our preliminary data show that all four approaches outlined above are feasible and they therefore offer realistic prospects for the design and implementation of anti-toxin strategies.
